1. Structural Basis for EGF Receptor Inhibition by the Therapeutic Antibody IMC-11F8 
Shiqing Li, Paul Kussie, Kathryn M. Ferguson 
Structure 
Volume 16, Issue 2, Pages (February 2008)
DOI: /j.str
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2. Figure 1 Ligand-Induced Dimerization of EGFR
In the unliganded state, EGFR exists as a tethered monomer (left). Domain II (green) interacts with domain IV (white, with secondary structure highlighted in green); domains I (white with red highlights) and III (red) are far apart. The arrangement of the domains in the ligand-induced, dimeric state (right) is dramatically different. Domains I and III are closer together and interact with the ligand (EGF, cyan). The colors of the right-hand molecule in the dimer are lightened for contrast. Domain IV in the dimer is modeled as described (Ferguson et al., 2003). The gray line represents the approximate location of the membrane. This figure uses coordinates from PDB ID codes 1YY9, 1NQL, and 1IVO.
Structure  , DOI: ( /j.str )
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3. Figure 2 Fab11F8 Binds to sEGFR and Inhibits EGF Binding to sEGFR
(A) SPR analysis of sEGFR binding to immobilized Fab11F8. A series of sEGFR samples of the indicated concentrations was passed over a Biosensor surface to which Fab11F8 had been covalently coupled. A representative data set of the equilibrium SPR response for each sample, expressed as the fraction of the maximum binding, is plotted as a function of the concentration of sEGFR. The inset shows that no additional binding is seen at higher sEGFR concentrations. The curve indicates the fit to a simple one-site Langmuir binding equation for the data set shown. A KD value of 3.3 ± 0.5 nM was obtained from at least three independent binding experiments.
(B) The ability of Fab11F8 to compete for sEGFR binding to immobilized EGF is shown. The indicated molar excesses of Fab were added to samples of fixed concentration of sEGFR (600 nM), and these samples were passed over immobilized EGF. The equilibrium SPR responses obtained for each sample, expressed as a fraction of the response with no added Fab, is plotted as a function of the molar excess of Fab. Error bars indicate the standard deviation of at least three independent measurements. All binding is abolished at a 1:1 stoichiometry of Fab11F8/sEGFR and the IC50 value for these conditions is 350 nM.
Structure  , DOI: ( /j.str )
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4. Figure 3 Fab11F8 Binds to Domain III of sEGFR
(A) Cartoon of the Fab11F8/sEGFR complex. The light chain of Fab11F8 is shown in yellow, and the heavy chain in orange. Domains of sEGFR are colored as in Figure 1.
(B) SPR analysis of sEGFRd3 binding to immobilized Fab11F8 performed and analyzed as described in Figure 2A. A KD value of 1.0 ± 0.1 nM was obtained.
(C) Cartoon of the Fab11F8/sEGFRd3 complex in the same orientation and colors as in (A).
(D) Detailed view of the Fab11F8/sEGFRd3 interface, rotated ≈30° about a horizontal axis with respect to (C). Secondary structure is highlighted in yellow for VL and orange for VH. The parts of the CDRs that interact with domain III are yellow for L1 and L3, cyan for H1, and orange for H2 and H3. Side chains that make direct hydrogen bond or key van der Waals contacts are shown in stick representation in colors as for the interacting CDRs. The main chain of domain III of sEGFR is show in a gray cartoon highlighted in red. Side chains on domain III that make key contacts with the Fab are shown in green stick representation and labeled in black. A transparent molecular surface is show around domain III. The darker gray shading indicates the region of this surface that is occluded from solvent by the interaction with Fab11F8, defined using the program CNS (Brunger et al., 1998).
Structure  , DOI: ( /j.str )
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5. Figure 4
Features of the Shared Fab11F8, FabC225, and EGF Binding Region on Domain III
(A) Molecular surface representations of domain III of sEGFR with the contact regions (as defined in Figure 3D) colored red for Fab11F8, yellow for FabC225, and blue for EGF. Orientation is looking down onto the domain III binding site.
(B) Functional features of the domain III molecular surface. In the left panel, the surface is colored by atom type: negative, red; positive, blue; polar oxygen, pink; polar nitrogen, light blue; and apolar, white. The right panel shows the electrostatic potential from −2.5 kT (red) to +2.5 kT (blue) projected onto the surface. Electrostatic potential calculations used the adaptive Poisson-Boltzmann solver (APBS) implemented in PyMOL (Baker et al., 2001; DeLano, 2004).
(C) Orthogonal views of domain III. High-mannose chains (yellow) have been placed at each position of glycosylation on sEGFR guided by the one or two ordered sugar groups that are seen in the X-ray crystal structures. For reference, the contact region of Fab11F8 is shown (red).
(D) Three orientations of sEGFR are shown with the electrostatic potential, as in (B), projected on the surface and high-mannose chains shown. Both electrostatic and carbohydrate steering might play a role in guiding Fabs or ligands to the shared binding site on domain III.
Structure  , DOI: ( /j.str )
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6. Figure 5
Fab11F8 and FabC225 Use Distinct Interactions to Recognize a Common Epitope
(A and B) Detailed view of the interactions of domain III of sEGFR with Fab11F8 (A) and FabC225 (B). The orientation is as in Figure 4A. Only those parts of Fabs that are involved in binding are shown. Interacting CDRs are colored as in Figure 3D. Side chains from the Fab that interact with sEGFR are shown in stick representation and labeled using the same color scheme. The epitope region on sEGFR is shown in white cartoon. Side chains from sEGFRd3 that interact with the Fab are shown in stick representation in pink with black labels. The same set of side chains on the sEGFR are shown in both panels. For clarity, side chains that line the hydrophobic binding pocket on domain III (F412, A415, V417, and I438) are not labeled. Hydrogen bonds are shown with dotted black lines. Key water molecules are shown as green spheres. Side chains on sEGFRd3 that are altered in analysis of the binding site mutations (Figure 6) are ringed.
(C) Amino acid sequence alignment of the variable domains of Fab11F8 and FabC225. Only nonidentical amino acids are shown for FabC225, with a period indicating identity with Fab11F8 and a dash indicating a gap. Amino acids involved in interacting with domain III are highlighted using the same colors as in (A). A dot above every tenth amino acid indicates Chothia numbering that is used in the text and all figures (Chothia et al., 1986). Chothia-defined CDRs are underlined and Kabat-defined CDRs are indicated by bold text. In the submitted PDB files, and in Li et al. (2005), the Fab amino acids are numbered sequentially.
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7. Figure 6
Effect of Alterations in Domain III on the Affinity of sEGFR for Fab11F8, FabC225, and EGF
The fold change relative to wild-type sEGFR of the binding affinity for each indicated altered sEGFR to immobilized Fab11F8 (yellow), FabC225 (magenta), and EGF (black). KD values of wild-type sEGFR, determined as described in Figure 2, are 3.3 ± 0.5 nM (Fab11F8), 2.3 ± 0.5 nM (FabC225), and 130 ± 4 nM (EGF). Positive (upward) fold changes indicate higher-affinity binding, while negative (downward) fold changes indicate weaker binding. Error bars indicate the standard deviation for at least three separate measurements.
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8. Figure 7 Comparison of the Structures of Fab11F8 and FabC225
(A and B) The root-mean-square deviation (rmsd) of Cα positions between Fab11F8 and FabC225 for VL domains (A) and VH domains (B). Backbone atoms for each pair of domains were individually superimposed using the program SUPERPOSE (CCP4, 1994). The CDRs are marked and highlighted in yellow for VL and orange for VH.
(C) Cartoons of the variable domains of Fab11F8 and FabC225 looking up from the domain III binding site. Kabat CDRs are in dark gray. Key side chains in the paratope are shown in stick representation and colored as in Figure 3D.
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9. Figure 8
Mechanism of Inhibition of EGFR Activation by IMC-11F8 and by Cetuximab
(A) Cartoon model of Fab11F8 bound to sEGFR colored as in Figure 3A. Domain I and the N-terminal portion of domain II (gray) have been modeled using the coordinates from PDB ID code 1YY9.
(B) Cartoon of the FabC225/sEGFR complex (PDB ID code: 1YY9) colored as in (A).
(C) The mechanism of inhibition of ligand-induced dimerization and activation of EGFR for IMC-11F8 and cetuximab based on the structures presented here and in Li et al. (2005). The binding of the antibody to domain III of EGFR prevents ligand binding and might also sterically inhibit the conformational change that must occur for dimerization.
Structure  , DOI: ( /j.str )
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